The major hypothesis of this application is that the mammalian sperm nuclear matrix, and the epigenetic organization of the paternal DNA into loop domains by this structure, are essential for the paternal genome to participate in normal embryogenesis. Understanding the functional roles of this sperm DNA organization has been the long-term goal of this grant. We have demonstrated that mammalian sperm DNA is organized into loop domains that are attached at their bases to the protein skeleton of the sperm nucleus, the nuclear matrix, in a cell specific manner that changes during spermiogenesis and embryogenesis. Recently, we reported evidence that mouse oocytes injected with sperm nuclei with intact nuclear matrices develop into live births, but those injected with sperm nuclei with unstable nuclear matrices do not. These data led us to our current hypothesis that the sperm nuclear matrix and its organization of DNA into loop domains is essential for normal embryogenesis. DNA organization into loop domains in somatic cells functions in the control of transcription and DNA replication, and somatic cell nuclear matrices interact directly with several transcription factors. Therefore, one implication of our hypothesis is that the sperm nuclear matrix helps define which parts of the genome are replicated and transcribed first in the pronucleus. Maintaining this functional organization of the paternal genome has implications for clinical reproductive centers as well as for understanding the role of the paternal genome in development. We will test the hypothesis that the sperm nuclear matrix is essential for normal embryogenesis by three different approaches. First, we will test whether mouse sperm nuclei with disrupted DNA loop domain organization but intact DNA can participate in normal embryogenesis when injected into oocytes by ICSI. We have preliminary data to support that they do not. Second, we will test whether the embryo inherits the DNA loop domain organization, directly, using FISH of on pronuclei, and early embryonic cells. Finally, we will test whether the sperm nuclear matrix attracts transcription factors to the male pronucleus after fertilization.